The overarching goal of this research project is to elucidate at a structural and mechanistic level critical features of both normal and pathogenic Notch signal transduction. Prior to activation, Notch is quiescent and resistant to proteolysis, bu ligand binding triggers the on state by stimulating proteolytic cleavage of Notch by the ADAM10 (or ADAM17) metalloprotease at a juxtamembrane site immediately external to the membrane. The work proposed here will address key unresolved questions in the Notch field, including: i) how Notch receptors specifically recognize their ligands, and ii) how ADAM10 proteolysis of Notch is achieved and regulated. We propose to address these key questions in signaling by pursuing the following specific aims: 1. Determine the basis for specific binding between Notch receptors and their canonical ligands. In this aim, we will combine structural, biochemical, and cell-based assays to address one of the central unresolved questions in Notch signal transduction: how do Notch receptors recognize their ligands? The top priority will be to solve an X-ray structure of a receptor-ligand complex. I the process of pursuing this highest-impact, long-term objective, we will determine the intrinsic selectivity of various Notch receptors for different Delta-like ligands, as well as determine structures of individual receptor and ligand fragments in isolation and/or in combination with blocking antibodies of potential therapeutic relevance. 2. Determine the basis for catalytic specificity of ADAM-family metalloproteases in ligand- dependent proteolysis of Notch receptors. Top priorities of this aim will be to identify the processing sites of the different Notch isoforms, to elucidate the structure of the full ADAM10 ectodomain, and to determine the role of the ADAM regulatory region on Notch proteolysis by comparing the enzymatic activities of the isolated protease domain and the full ectodomain on Notch-derived substrates. Successful completion of these aims will constitute a major breakthrough in our understanding of this fundamental signaling pathway and the role it plays during normal development and in the pathophysiology of diseases associated with aberrant Notch signaling. By deepening our knowledge about the structural and biochemical foundations underlying ligand engagement and regulated proteolysis, our studies will identify new therapeutic strategies for management of Notch-related human pathologies, which include developmental disorders, neurodegenerative diseases, cardiovascular diseases, and cancer.